Prevalence of CYP2C19 Genetic Polymorphism among Normal People and Patients with Hepatic Diseases.

Background
Patients with hepatic diseases are treated with numerous drugs metabolized by cytochrome P450.


Objective
To evaluate the frequencies of CYP2C19 variant alleles (*2, *3, and *17), genotypes, and phenotypes, and the relationship between the frequency of these alleles and the underlying hepatic diseases among patients with advanced liver diseases who were candidates for liver transplantation.


Methods
The Study was conducted on 120 patients suffering from various hepatic disorders, candidates for liver transplantation, and 52 healthy volunteers. DNA was extracted from blood samples and analyzed by TaqMan SNP genotyping assay. The CYP2C19 genotypes were classified into poor, extensive, intermediate, and ultra-rapid metabolizer phenotypes.


Results
Viral hepatitis was the most common cause of liver disease among studied patients. The frequencies of CYP2C19 alleles *1, *17, and *2 were 66.7% (160/240), 20.8% (50/240) and 12.5% (30/240), respectively. Allele CYP2C19*3 was not found in the studied population. The most prevalent genotypes were CYP2C19 *1/*1 (47.5%) and *1/*17 (24.2%). The predicted CYP2C19 phenotypes were extensive metabolizer (47.5%), heterozygote extensive metabolizer (45.9%), ultra-rapid metabolizer (5%), and poor metabolizer (1.6%). There was no significant difference between the frequencies of CYP2C19 genotypes between healthy people and patients. The distribution of CYP2C19 genotype frequencies was not significantly associated with the underlying disease conditions (p=0.472).


Conclusion
The distribution of CYP2C19 genotype frequencies in Iranian healthy people and patients with various hepatic diseases was not significantly different. This may allow the physicians to predict a tailoring dose regimens based on the individual's metabolic capacity, decrease the risk of harmful side effects of the drugs, and optimize the treatment.


INTRODUCTION
G enetic polymorphism of drug-metabolizing enzymes is mostly associated with inter-individual and inter-ethnic variations in the pharmacokinetic and pharmacodynamic responses to drugs. This may influence the drug action and lead to differences in the efficacy or toxicity of pharmacological agents used [1]. Among several genes responsible for drug metabolism, cytochrome P450 genes have an important role in encoding many enzymes involved in the metabolism of a wide variety of drugs [2]. The hepatic cytochrome P450 2C19 (CYP2C19) is one of the main isoforms of cytochrome P450 2C subfamily, represented by related genes located on chromosome 10 [3,4]. Many variant alleles have been identified for CYP2C19, as a highly polymorphic gene. The wild-type CYP2C19*1 allele is the completely functional form of the CYP2C19 identified [5]. The most frequent CYP2C19 loss-of-function polymorphic alleles are two defective variants including *2 (rs4244285) with single-base substitutions (681G>A), resulting in a splic-ing defect and *3 (rs4986893) with a point mutation (636G>A), producing a premature stop codon [6]. CYP2C19*2 and *3 are the variant alleles with insufficient activity that can cause null function of the enzyme [6]. The other CYP2C19 gene variant is *17 (rs-12248560), located in a single nucleotide polymorphism (SNP, 806C>T) in a transcriptional regulatory region of the gene, leading to the enhancement of the transcriptional activity of the enzyme and ultra-rapid metabolism of CYP2C19 substrates [7]. Various combinations of these alleles lead to different classes of CYP2C19 phenotypes: poor metabolizer (PM, CYP2C19*2*2 and *3*3), intermediate metabolizer (HEM, heterozygote extensive matabolizer, CYP2C19*1/*2, CYP2C19*1/*17, CYP2C19*2/*17), extensive metabolizer (EM, CYP2C19*1/*1), and ultra-rapid metabolizer (URM, CYP2C19*17*17) [6,7]. Various studies have shown great ethnic differences in the frequency of PM phenotypes, ranging from 3% to 5% in Caucasians, 12% to 23% in Orientals [8], and 4% to 8% in Africans [9]. The frequency of URM was reported to be 18% to 26% in Caucasian [10], and 0.4% to 1.4% in Asian populations [11].
The efficacy and safety of pharmacogenetic drugs are related to the gene frequencies in different populations. Patients suffering from hepatic disorders and candidates for liver transplantation use numerous drugs, the metabolisms of which depend on the cytochrome P450 activity. The objective of this study was to evaluate the frequencies of CYP2C19 mutant alleles (*2, *3, and *17), genotypes, and phenotypes, and the relationship between the frequency of these alleles and the underlying hepatic diseases among patients with advanced liver diseases who were candidates for liver transplantation.

PATIENTS AND METHODS
During the patients follow-up, 3 mL of EDTA blood samples were taken from 120 patients who were candidates for liver transplantation and who referred to the Transplant Center in Nemazee Hospital, Shiraz, Iran, from April

DISCUSSION
Having compared CYP2C19 alleles and genotypes frequencies in patients with advanced liver diseases and healthy individuals revealed no significant difference. Also, the CYP2C19 genotype frequencies were not significantly different among patients with various underlying hepatic diseases. Patients with hepatic disorders receive numerous drugs to treat their complications including both hepaticand non-hepatic-related disorders. The best known medications are proton pump inhibitors (PPIs), antivirals, antifungals, anti-depressants, immunosuppressives, beta blockers, and pain killers. Some of these commonly prescribed drugs are metabolized by CYP2C19 [12]. Effective serum level of PPIs is needed in patients with hepatic failure, suffering from esophageal varices and gasterointestinal complications associated with drugs consumption. The lowest plasma concentration of omeprazole and 19-fold higher, were reported in URMs and PMs after similar dose of PPIs treatment by Payan, et al [13]. Nazir, et al, showed a significant rise of omeprazole serum concentration in PMs, compared with EMs indicating that lower doses of PPIs are required in PMs to reach the optimal concentrations and effects [14]. For the best management of these patients, knowledge about the CYP2C19 genotyping could be helpful to improve treatment efficacy and decreased adverse effects.
The allele frequencies of CYP2C19*1, *2, and *17 in our study were 66.7%, 12.5%, and 20.8%, respectively. Different results have been reported in other studies ( Table 3). The frequencies of the CYP2C19*2 alleles found in the present study population were close to Saudi Arabian, Greek, Egyptian, and Russian populations (10.9% to 13.0%), but lower than the frequencies in the Indians and Tamilians (29.7% and 37.9%, respectively) [15][16][17][18][19][20]. CY-P2C19*3 allele was absent in our study and other different populations such as Saudi Arabian, Greek, Indian, and Italian people [15,16,19,21]. Its frequency was reported to be 1% and 1.8% in Iran [22,23], 12.8% in Egypt, and 2.2% in Tamil [17,20]. The most frequently identified variant allele in the present study was *17 (20.8%). The available data on this allele are limited, but like other alleles, its distribution is different in each ethnic group. The CYP2C19*17 frequency in Saudi Arabia was reported to be 25.7% [15], in line with our results. However, it was 1.3% in Japanese [24], which is lower than our findings.
The CYP2C19*2*2 genotype frequency, as PM in our study, was 1.6%. This result was consistent with those reported by Payan, et al, (1.8%) and Azarpira, et al, (1.3%) among Iranian healthy individuals [13,22]. The PM genotype was reported to be 2.8% in patients with coronary artery disease [25]. The genotype frequencies of CYP2C19*2*2 among ethnicities are 2.1% in Greece, 1.7% in Russia, and 3.8% in Denmark [16,18,26]. The individual differences in CYP2C19 genotype status among these populations could be due to differences in the studied populations.
The distribution of frequencies of CYP2C19*1, *2, and *17 alleles in the present study followed the Hardy-Weinberg equilibrium. Being in Hardy-Weinberg equilibrium reflects a population with random mating and without immigration, mutations or natural selection (i.e., every individual has an equal chance of survival) [27]. The frequency of CYP2C19*3 was not in Hardy-Weinberg equilibrium in the present study. The small sample size of our study (one of its limitations) may lead to missed rare alleles.
The antidepressants, sertraline and escitalopram, are both metabolized by CYP2C19. Patients with defective CYP2C19 alleles (CY-P2C19*2 and *3) showed higher serum levels of sertraline than those with the wild-type allele (CYP2C9*1) after administration of the same dose of the drug [28].
Patients with advanced liver diseases have an increased susceptibility to bacterial and fungal infections due to receiving prolonged courses of corticosteroids. The mortality rates of invasive aspergillosis in liver transplant and patients with hepatic disorders were reported 20% and 63.2%, respectively [29,30]. Voriconazole is an effetive antifungal agent for the treatment of such infections [31,32]. Several studies have shown the potential correlations of CYP2C19*2, *3 defective alleles with higher and CYP2C19*17 allele with lower serum voriconazole level leading to adverse events or therapeutic failure [33][34][35].
A wide variety of other medications including antiepileptic (phenytoin and carbamazepine), antidepressant (imipramine), antivirals (nelfinavir), and tuberculostatics (isoniazide and rifampin), extensively prescribed for patients with advanced liver diseases, are the substrate, inducer or inhibitor of CYP2C19 [36,37]. There are potential risks of toxicity associated with use of such drugs and change in their metabolism as a result of CYP2C19 polymorphism.
In conclusion, our assessment in patients with hepatic failure and healthy individuals revealed no significant difference in the frequencies of CYP2C19 genotypes. Therefore, management of this population is the same as normal population. This may allow the physicians to predict a tailoring dose regimens based on the individual's metabolic capacity, to decrease the risk of harmful side effects of the drugs and optimize the treatment.

ACKNOWLEDGEMENTS
This work was based on a PhD thesis by Zahra Hashemizadeh as a requirement for graduation in PhD of Mycology from Prof. Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran. The study was financially supported by a Grant (92-6884) from Shiraz University of Medical Sciences, Shiraz, Iran. Our thanks go to H. Khajehei, PhD, for copyediting of the manuscript.

CONFLICTS OF INTEREST:
None declared.